The project objective is to identify and characterize genes involved in bacterial cell differentiation. Vegetative cells of the filamentous cyanobacterium Nostoc punctiforme (strain ATCC 29133) are capable of cellular differentiation into spore-like akinetes that function in perennation. N. punctiforme is an excellent model systems in which to study cellular differentiation with respect to its recently sequenced genome and well developed genetic system. The first specific aim of this proposal is to identify genes differentially expressed during akinete differentiation. This study will utilize a glucome-6-phosphate dehydrogenase mutant strain of N. punctiforme, designated UCD341, that differentiates a large proportion of vegetative cells into akinetes following dark incubation in the presence of fructose. The wild-type strain is capable of heterotrophic growth under similar conditions and does not differentiate into akinetes. The differential display technique will be used in conjunction with the sequenced genome to identify genes induced or repressed in akinetes. The second specific aim is to construct and physiologically6 characterized mutants in selected differentially expressed genes by targeted gene replacement. Transcriptional reporter constructs will be generated for selected genes and introduced into the wild-type and UCD341 strains to determine temporal and spatial (cell- type specific) expression patterns. Promoter sequences from differentially expressed genes will be determined by primer extension and analyzed by sequence comparison for common promoter elements. Functional characterization of differentially expressed gene products in akinetes will lead to a better physiological understanding of this cell type, and form the basis for addressing the long-term of identifying regulatory mechanisms involved in akinete differentiation.